The present invention relates to the growth of human epidermal cells to produce useful sheet-like products, the resultant products, and use thereof, for example, as skin graft materials.
More particularly, the present invention, and certain of its process embodiments, relate in part to the dissociation of epidermis into epidermal cells and the growth of the epidermal cells into a layer of epidermis consisting essentially of epidermal cells which do not express immune stimulatory antigens. Another aspect of the present invention involves sheet-like products comprising at least one layer of epidermis grown in culture and consisting essentially of non-immune-competent epidermal cells and the use thereof as a skin grafting material.
Previously, the present inventor and a co-worker at that time jointly developed a process for growing human epidermal cells to form an epidermal sheet. See U.S. Pat. No. 4,254,226, naming Eisinger and Hefton as co-inventors. U.S. Pat. No. 4,254,226 was filed as a continuation application of subsequently abandoned parent application Ser. No. 749. U.S. Pat. No. 4,299,819, naming Eisinger as sole inventor and claiming the use of the Eisinger and Hefton epidermal sheet in the treatment of burn victims, was filed as a divisional application of Ser. No. 749. Work related to the Eisinger and Hefton process and epidermal sheet product is found described in Eisinger et al, "Human Epidermal Cell Cultures: Growth and Differentiation in the Absence of Dermal Components or Medium Supplements," Proc. Natl. Acad. Sci. USA, volume 76, No. 10, pp. 5340-5344 (October, 1979). Also, see Eisinger et al, "Wound Coverage by a Sheet of Epidermal Cells Grown In Vitro from Dispersed Single Cell Preparations," Surgery, St. Louis, Volume 88, No. 2, pp. 287-293 (August, 1980).
The above described prior art process of Eisinger and Hefton may be summarized as broadly involving the sequence of separating the epidermis in human skin from the dermis, dissociating the epidermis into epidermal cells and growing the epidermal cells in a tissue culture medium. Specific process conditions are disclosed by Eisinger et al for use in each of the above steps and certain of these process conditions will be discussed in greater detail hereinbelow in relationship to a corresponding proces step in the cell growth process embodiments of the present invention.
Although there are a number of prior art processes, such as that of Eisinger and Hefton, for growing epidermal cells, to a significant extent the resultant products have been rejected when used as a skin graft for a recipient unrelated to the precursor cell donor, the reason being the rejection of the skin graft cells by the recipient because of the presence of transplantation immunogens on sub-populations of the human epidermal cells of the grown tissue. The transplantation immunogens are present on the Langerhans cells and other cell sub-populations, the latter probably being dendritic epidermal cells. In the above-cited reference, Eisinger 1979 Proc. Natl. Acad. Sci. article, ATPase staining was used as an analytical tool to determine the presence of immune-competent Langerhans cells, which are included in the cells carrying surface antigens which cause transplantation rejection. The products of Eisinger and Hefton contain 2 to 3% ATPase positive cells.
Morhenn et al, "Cultured Human Epidermal Cells Do Not Synthesize HLA-Dr", The Journal of Investigative Dermatology, 78:32-37 (1982) state that they obtained epidermal cell cultures grown on collagen gel or gelatin membranes free of the immune stimulatory antigen (HLA-Dr) and also free of Langerhans cells, following a culturing period of 7 days or longer. It is the present inventor's belief that the absence of HLA-Dr immunogens in Morhenn's product is due to a combination of culturing parameters, probably including the use of the collagen or gelatin substrate and not due solely to the duration of culturing. The present inventor has carried out culturing of epidermal cells for lengths of time of the same order as Morhenn et al and has found the HLA-Dr transplantation immunogens to be present. Furthermore, Morhenn et al (1) state that their cultured epidermal cells include some ATPase positive cells, which would be expected to be Langerhans cells, but if not could be a type of non-Langerhans dendritic cell possessing some immunocompetence, and (2) do not describe a process for producing an integral self-supporting sheet consisting of epidermal cells. The composite structure of Morhenn, including the collagen or gelatin substrate, would in itself be poorly suitable as a skin graft.
Earlier work of the present inventor has been abstracted by Hefton et al, "Guinea Pig Epidermal Cell Cultures: Development of Confluent Sheets and Their Transplantation," Federal Proceedings, Volume 39:736 (1980) where transplantation was onto syngenetic animals and by Hefton et al, "Human Epidermal Cells No Longer Stimulate Allogeneic Lymphocytes after Growth in Culture," J. of Investigative Dermatology, Volume 76:308 (1981) where the cultured cell population still contained a low percentage of cells expressing HLA-Dr antigens.